Polymerases are members of a class of template dependent enzymes that can catalyze the growth of a chain of nucleotides. Depending on the configuration of the active site or sites within a polymerase, the enzyme may have a polymerization functionality that imparts the ability to join together two ribonucleotide units, two deoxyribonucleotide units or a ribonucleotide unit and a deoxyribonucleotide unit. By way of example, a DNA dependent DNA polymerase utilizes a DNA template and produces a DNA strand that is complementary to at least a portion of a DNA template. An RNA-dependant DNA polymerase, e.g., a reverse transcriptase, uses an RNA template to produce a DNA strand that is complementary to at least a portion of an RNA template. Examples of known polymerases include DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase α, DNA polymerase β, DNA polymerase γ, DNA polymerase δ and DNA polymerase ε.
As persons of ordinary skill in the art are aware, when joining two nucleotides together, or adding a nucleotide to a growing chain of nucleotides, polymerases rely on a template strand to determine what should be the next nucleotide in the growing chain. Thus, they read the template strand but write the opposite strand.
By convention, individual nucleotides are described as having 5′ and 3′ positions, and in a chain the 3′ position of one nucleotide is linked to the 5′ position of the next nucleotide. Because each nucleotide has a 5′ end and a 3′ end, each strand of nucleotides is referred to as having a 5′ end, which refers to the position at the end of the strand that has a nucleotide with a 5′ position that is not bound to another nucleotide, and a 3′ end, which refers to the position at the end of the strand that has a nucleotide with a 3′ position that is not bound to another nucleotide.
Polymerases grow a chain from the 3′ end, and they add the next nucleotide by joining the 5′ end of the nucleotide to be added to the 3′ end of the chain. Thus, polymerases are said to write in a 5′→3′ direction.
When adding nucleotides to a growing chain, polymerases prefer to obey the Watson-Crick rules for base pairing, i.e., when a DNA or RNA polymerase is used and there is a C on a template strand, preferably on the growing strand it should incorporate a G; when a DNA or RNA polymerase is used and there is a G on a template strand, on the growing strand it should incorporate a C; when a DNA polymerase is used and there is an A on a template strand, on the growing strand it should incorporate a T; when a DNA polymerase is used and there is a T or a U on a template strand, on the growing strand it should incorporate an A; when an RNA polymerase is used and there is an A on a template strand, on the growing strand it should incorporate a U; and when an RNA polymerase is used and there is a U or a T on a template strand, on the growing strand it should incorporate an A.
Polymerases may have multiple functionalities. For example, certain polymerases also have an exonuclease functionality in addition to a polymerizing functionality. An exonuclease functionality refers to the ability of the polymerase to remove nucleotides. Some polymerases may have either a 3′→5′ exonuclease functionality, or a 5′→3′ exonuclease functionality, or both functionalities, or neither functionality. The phrase “exonuclease functionality” refers to the ability to remove a nucleotide that the enzyme, depending on its identity and the reaction conditions, determines is located in a place that it should not be, which may, for example, be due to there being a mismatch or a modification to the nucleotide, or a nucleotide being ahead of a growing chain and incompatible with the current chain growth. Thus, if the exonuclease is a 5′→3′ exonuclease, it removes nucleotides that are in front of it as it moves in the 3′ direction and grows a chain of nucleotides. A polymerase with 5′→3′ exonuclease activity displaces a few bases from the 5′ end and cleaves one or a few bases from the displaced end. Thus, the 5′→3′ activity is also an endonuclease. An example of a DNA polymerase is Finnzymes' Native thermostable DyNAzyme™ I DNA Polymerase. That enzyme is isolated and purified from Thermus brockianus, which is a Finnzymes' proprietary strain. It contains 5′→3′ exonuclease functionality. In contrast, 3′→5′ exonuclease functionality refers to the ability to remove nucleotides from the 3′ terminus of a nucleic acid.
Polymerases are well known tools for use in applications that seek to sequence the content of the genome or sections of it. Polymerases with altered functionalities have been used in DNA sequencing applications. One example is a method for sequencing DNA using a mixture of polymerases, one polymerase with a reduced ability to incorporate dideoxynucleotides (ddNTPs) into a synthesized DNA strand, the other polymerase allowing incorporation of ddNTPs into the synthesized strand, due to a Tabor-Richardson mutation in which tyrosine replaces phenylalanine in the polymerase crevice. During polymerization of the DNA being synthesized, the mutation is responsible for discriminating between incorporation of either deoxynucleotides or dideoxynucleotides. The result of using this polymerase mixture is an increased statistical probability that chain termination occurs due to dideoxynucleotide incorporation into the DNA being synthesized, which permits amplification and sequencing reactions to be performed with fewer manipulations (U.S. Pat. No. 6,225,092). Another example is a mixtures of polymerases used in DNA sequencing, DNA labeling, DNA amplification, cDNA synthesis reactions, or analyzing and/or typing polymorphic DNA fragments. The polymerase mixture contains a polymerase having 5′→3′ exonuclease activity (exo+) and a polymerase having reduced 5′→3′ exonuclease activity (exo−), where the presence of the exo− polymerase enhances PCR performance by increasing the amount of product produced (U.S. Patent Application Publication No. 20060292578). Because these methods to analyze and/or type polymorphic DNA fragments compare the size or the sequence of the resulting amplified fragments, enhanced PCR performance facilitate such size comparisons or the ability to sequence an amplified fragment from each individual. Another example is use of blends of chimeric and non-chimeric thermostable DNA polymerases in DNA sequencing, where the blend of chimeric and non-chimeric polymerases allows PCR reactions with shorter extension times that facilitate PCR amplification of genomic DNA templates and improve efficacy of long PCR.
Polymerases can be used in combination with a hydrolysis probe assay which may, e.g., rely on Taqman® chemistry. In these hydrolysis probe assays, the polymerase with 5′→3′ exonuclease activity, e.g., Taq polymerase from Thermus aquaticus, can catalyze the growth of a chain of oligonucleotides and also degrade from the 5′ direction, a hybridized non-extendible DNA probe during the PCR extension step. The probe that is degraded is designed to hybridize to a region within the amplicon and is dually labeled with a reporter dye and a quencher dye. The close proximity of the quencher dye to the reporter dye suppresses the fluorescence of the reporter dye. During amplification and in the presence of non-terminating nucleotides and primers that span the probe site, the polymerase will grow a strand until it gets to the probe. Next it will excise the probe from the 5′ end, thereby degrading it. After the exonuclease activity of the polymerase degrades the probe, the reporter dye fluorescence increases because the reporter dye is not quenched by the quencher dye. Fluorescence increase is proportional to the number of probes cleaved, and thus is also proportional to the amount of template present.
Some variants of hydrolysis probe assays rely on a 3′→5′ exonuclease activity, e.g. proofreading activity, to generate a signal. For example, U.S. Pat. No. 7,163,790 describes an “error-correcting assay” using an oligonucleotide probe labeled on the 3′ nucleotide, and a polymerase having 3′→5′ exonuclease activity, referred to as a “error-correcting polymerase”. In this error correcting assay, if there is a match between the labeled 3′ nucleotide of the probe and the target nucleic acid, e.g., if the 3′ nucleotide anneals to the target nucleic acid, the labeled 3′ nucleotide is not accessible to the 3′→5′ exonuclease activity of the error correcting polymerase. If, however, there is a mismatch between the labeled 3′ nucleotide and the target nucleic acid, the labeled 3′ nucleotide is accessible to the 3′→5′ exonuclease activity of the error correcting polymerase and is cleaved. Cleavage of the labeled 3′ nucleotide is then detected, e.g., by a decrease in fluorescence polarization. This error correction method relying on 3′ labeling and 3′→5′ exonuclease activity has been expanded to include methods for quantifying nucleic acid amplification using unlabeled primers (U.S. Patent Application Publication No. 20060024695). U.S. Pat. No. 7,445,898 describes use of polymerases and/or enzymes having 3′→5′ exonuclease activity, where the ratio of double stranded (ds) to single stranded (ss) 3′→5′ exonuclease activity is optimized to result in greater label cleavage from the 3′ end of a labeled probe, and therefore greater signal.
One variation of hydrolysis probe assay chemistry is an assay for allelic discrimination. For example, humans have two copies of the genome in each cell. These copies are not exactly the same and have many types of sequence variations. One of these variation types is the single nucleotide polymorphism (SNP), in which the nucleotide base sequence has a single nucleotide difference from the normal type. When both copies of the genome sequence are the same for a certain region, the individual may be referred to as homozygous. When the copies are different in this region, the individual may be referred to as heterozygous. When both copies represent the normal type, the individual may be referred to as being a homozygous wild type. When both copies represent the same difference from the normal type, the individual may be referred to as being a homozygous mutant. For typical variations in the genome there are three different genotypes: homozygous wild type, homozygous mutant, and heterozygous. Hydrolysis assays can be used for discriminating these types. The typical discrimination assay contains two primers that define the region that contains the variation of interest to be amplified, and two probes, each specific for one type of sequence variant and labeled with different reporter fluorophores. In the homozygous sample, only one type of the probe hybridizes and is cleaved to produce a signal. In the heterozygous sample, both probes bind and produce a signal. The sample genotype is determined from the fluorescence data by comparing the intensity changes of each probe before and after the PCR reaction or in each cycle in real time qPCR.
Unfortunately, in these applications there can be unacceptably high levels of incorrect or ambiguous results. Under common conditions, there may be quick and efficient cleavage of any bound probe, which includes a probe with a mismatch that binds transiently to the target sequence and thus is present long enough for the exonuclease activity to occur. This false positive can lead to fluorescence increases even when cleavage should not have occurred, thereby leading to poor allelic discrimination due to a high mismatch signal. This in turn may cause an incorrect sequence or allele to be inferred, or there may be an unacceptable level of ambiguity. These undesirable results are unfortunately too common when probes differ by a few nucleotides or only one nucleotide, as is the case with single nucleotide polymorphisms. Thus, there is a need to improve hydrolysis probe assay systems used for allelic discrimination and applications in which the mixtures are used.